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WO1998035687A1 - Phragmoplastine et methodes d'examen du developpement de plaques cellulaires - Google Patents

Phragmoplastine et methodes d'examen du developpement de plaques cellulaires Download PDF

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WO1998035687A1
WO1998035687A1 PCT/US1998/002858 US9802858W WO9835687A1 WO 1998035687 A1 WO1998035687 A1 WO 1998035687A1 US 9802858 W US9802858 W US 9802858W WO 9835687 A1 WO9835687 A1 WO 9835687A1
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phragmoplastin
protein
cells
cell
cell plate
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WO1998035687A9 (fr
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Desh Pal S. Verma
Xiangju Gu
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Ohio State University Research Foundation
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5097—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving plant cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the cell plate is a disc-like structure that is present only m dividing plant cells Since cell plates are found only m plant cells, it is hoped that herbicides which directly and specifically block or interfere with cell plate development will be specific to plants and less hazardous to animals that may be exposed to such herbicides Unfortunately, identifying such herbicides is hampered by a scarcity of tools and methods for rapidly and easily monitoring the effect of such herbicides on cell plate development
  • the present invention provides new tools an methods for identifying herbicides and potential herbicides which affect cel_ plate formation and development
  • a new protein, phragmoplastin na ⁇ been discovered which is associated w th cell plate membrane vesicles during cytokinesis
  • visualizing the phragmoplastin one is able to examine the development of the cell plate particularly m response to herbicides or potential herbicides
  • One method of visualizing phragmoplastin employs immunocytochemical techniques with anti- phragmoplastm antibodies.
  • Another method of visualizing phragmoplastin employs cells which are transformed with a DNA molecule which encodes a chimeric phragmoplastin protein comprised of phragmoplastin and a luminescent tag or protein, fused to the phragmoplastin protein.
  • the phragmoplastin m such cells is visualized by examining the cells under conditions which cause the marker to become visible such as by fluorescent microscopy.
  • the present invention also relates to isolated DNA molecules which encode phragmoplastin, and cells transformed with DNA which encodes phragmoplastin, particularly DNA which encodes chimeric phragmoplastin proteins.
  • Figure 1 is a nucleotide sequence, Seq. ID No . 3, of the phragmoplastm-encodmg cDNA of plasmid pPDL5 ;
  • Figure 2 is a nucleotide sequence, Seq. ID. No. 5, of the phragmoplastm-encodmg cDNA of plasmid pPDL12 ;
  • Figure 3 is the predicted ammo acid sequence, Seq. ID No. 4, of the phragmoplastin encoded by the cDNA of plasmid pPD 5 as shown m Figure 1 ;
  • Figure 4 is the predicted ammo acid sequence, Seq. ID No 6, of the phragmoplastin encoded by the cDNA of plasmid pPDL12 as shown Figure 2 ;
  • FIG. 5 is a map of plasmid pBI121-EGP. Detailed Description of the Invention
  • a new protein, phragmoplastin which is spatially and temporally associated with the cell plate membrane during all stages of cell plate development has been discovered.
  • visualizing the phragmoplastin one is able to examine the development of the cell plate particularly m response to herbicides or potential herbicides.
  • the present invention relates to different methods of examining the effect of potential herbicides on cell plate development.
  • growing plant cells are treated with the herbicide .
  • immunocytochemical methods which employ anti-phragmoplastm antibodies are used to visualize the distribution of the phragmoplastin withm the cell and to determine whether the herbicide inhibits formation or development of the cell plate.
  • the herbicide is administered to living transgenic plant cells comprising a DNA molecule which encodes the chimeric phragmoplastin protein.
  • the effect of the herbicide on cell plate formation and development is determined by microscopy to visualize the distribution of the chimeric protein m transgenic cells that have been treated with the herbicide.
  • phragmoplastin has a calculated molecular weight of about 68 3 kDa
  • the phragmoplastin proteins depicted m SEQ. ID. NOS. 4 and 6 have three consensus sequences which are known to be involved m GTP binding • the Gl region G(X) 4 GK(ST) at position 41-48, the G3 region DX 2 G at position 142-145; and the G4 region (TN) (KQ)XD at position 211-214. All three GTP binding sequences are the N-terminus region of phragmoplastin.
  • the N-terminus of the phragmoplastin also comprises a self-assembly motif, which includes ammo acid residues 49-93 of the phragmoplastin proteins depicted m SEQ. ID NOS. 4 and 6.
  • the phragmoplastin proteins also have a putative calcium/calmodulm- dependent protein kmase and cAMP-dependent protein kmase A site in the C-terminus, which is located at position 574 of the phragmoplastin proteins depicted m SEQ. ID.NOS. 4 and 6.
  • Phragmoplastin has a 41 6% homology with the animal protein dynamm. Phragmoplastin, however, lacks the C-termmal prol e-rich domain of dynamm. Phragmoplastin also lacks a transmembrane binding domain as found m dynamm.
  • phragmoplastin as used herein includes proteins which comprise the ammo acid sequence of Figure 3 and SEQ. ID. NO 4 and the ammo acid sequence of Figure 4 and SEQ. ID. NO.6 Phragmoplastin also includes proteins which have conservative ammo acid substitutions m the sequences of SEQ. D.NOS 4 and 6, proteins which are allelic variants of proteins comprising the ammo acid sequences of SEQ. ID. NOS 4 and 6, and proteins comprising ammo acid sequences that are 80% homologous, preferably 90% homologous, more preferably 95% homologous to the sequences of SEQ. ID. NO 4 or SEQ. ID. NO. 6.
  • phragmoplastin is localized m the permuclear region of meristemic plant cells and is not concentrated any specific areas during the early stages of the cell cycle During early anaphase, when cell plate formation begins, phragmoplastin redistributes to the equator of the cell, where it is present across the entire cell plate disc within the phragmoplast cylinder.
  • phragmoplastin redistributes to the growing edge of the cell plate
  • phragmoplastin plays a role cell plate development and cell wall formation.
  • phragmoplastin is a useful marker for cell plate formation and development m growing plant cells. Accordingly, antibodies to phragmoplastin are useful reagents for monitoring the effect of herbicides on cell wall formation and cell wall development.
  • transformed cells that comprise a DNA molecule which encodes a chimeric protein comprising the phragmoplastin protein fused to a luminescent peptide or protein tag are also useful for monitoring the effect of herbicides on cell plate development m living cells. Such cell lines are particularly useful for screening for herbicides that bind to phragmoplastin or that specifically block redistribution of phragmoplastin m the cell.
  • phragmoplastin is prepared using standard recombmant DNA methodology.
  • Such methodology typically involves introducing a polynucleotide, preferably a DNA molecule, which encodes the phragmoplastin protein into a host cell.
  • the phragmoplastin encoding sequences is expressed m the host cell such that phragmoplastin protein molecules are formed m the cell
  • the introduced polynucleotide also comprises a promoter, more preferably an ducible promoter, operably linked to the phragmoplastin encoding sequence
  • the polynucleotide further comprises a sequence which encodes a tag to facilitate isolation of the phragmoplastin protein such as, for example, an affinity tag and/or an epitope tag.
  • the tag sequences are at the 5 'or 3' end of the phragmoplastin-encoding sequence.
  • the polynucleotide preferably also comprises nucleotide sequences that encode a replication origin and a selectable marker.
  • the polynucleotide comprising the phragmoplastin-encoding sequence is introduced into the host cell by conventional methods, such as, by cloning the polynucleotide into a vector and by introducing the vector into the host cell by conventional methods .
  • Suitable host cells include , for example, bacterial cells, yeast cells, mammalian cells, and plant cells.
  • the DNA sequences of the introduced polynucleotide are then expressed in the host cell. Thereafter the expressed protein is isolated from the host cell using conventional methods such as for example, chromatography and gel electrophoresis .
  • phragmoplastin can also be prepared using conventional synthetic biochemical techniques, such as solid phase peptide synthesis.
  • phragmoplastin and anti-phragmoplastin antibodies were prepared using the following procedures. Cloning- cDNA molecules which encode phracrmoplastin protein from dividing plant cells. cDNA clones which encode phragmoplastin were obtained using the following primers :
  • soybean nodule poly (A) RNA that had been isolated from 15-day- old root nodules using oligo (dT) .
  • the isolated poly (A) RNA was then reverse transcribed into cDNA using oligo (dT) .
  • PCR amplification of the resulting product was performed as 45 cycles at 94°C for 40 seconds; 55°C for 40 seconds; and 72°C for 1 minutes
  • the resulting 500 base pair fragment obtained was blunt-end ligated into the Smal site of pUC19 and used as a probe to screen a ⁇ -ZapII soybean nodule cDNA library.
  • Four positive clones were identified after screening lxlO 6 plaques. The positive clones were excised and ligated into plasmids obtained from Stratagene and sequenced using the procedure of Sanger et al, 1977, m Proc . Nat. Acad Sci. USA 74.5463-5467.
  • the other clone, pPDL12 is encoded by a different gene.
  • the nucleotide sequence of the pPDL5 clone is shown Figure 1 and SEQ. ID. NO. 3.
  • the nucleotide sequence of the pPDL12 clone is shown m Figure 2 and SEQ. ID . NO . 5.
  • both pPDL5 and pPD 12 contain nearly full-length cDNA inserts with poly (A) tails and with 5'non- cod g regions of 126 base pairs (pPDL5) and 159 base pair (pPD 12) .
  • Each of these two nucleotide sequences contains a long open reading frame of 1930 base pairs corresponding to a protein of 610 ammo acids m length with a calculated molecular weight of 68.3 kDa
  • the two clones differ m the N-termmal and C-termmal non-coding regions, but share 98% homology m their coding regions.
  • Polynucleotide molecules which encode phragmoplastin protein include, for example, the nucleotide sequences of SEQ. ID. NO. 3 or SEQ. ID. NO.5 or nucleotide sequences which hybridize under stringent conditions to the sequences of SEQ. ID. NOS. 3 or 5.
  • stringent conditions means hybridization will occur if there is at least 90% identity between the polynucleotide sequence and one of SEQ. ID. NO.
  • the polynucleotide molecules which encode phragmoplastin include, for example, DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA, and RNA sequences encoding allelic variant forms of the peptides encoded by the cDNA of SEQ. ID. NOS. 3 and 5.
  • DNA or RNA is provided m a purified or isolated form.
  • the polynucleotide molecules which encode phragmoplastin optionally encode a marker sequence which allows for purification of the phragmoplastin, such as for example an affinity tag or epitope tag
  • the polynucleotide molecules which encode phragmoplastin optionally encode a sequence which encodes a luminescent peptide or protein fused to the coding sequence for phragmoplastin . Purification of Phracrmoplastin from E coli transformed with a cDNA molecule that encodes Phracrmoplastin.
  • the Bgr/II-Kpml fragment of pPDL12 was cloned m frame into the Barri ⁇ I -Kpnl site of bacterial expression vector pRSETC, which had been obtained from Invitrogen.
  • the resulting construct pEPDL-12 contains an lsopropyl-B-D-thiogalactopyranoside (IPTG) -regulatable promoter operably linked to a coding sequence for a polyhistidme metal binding domain fused to the coding sequence of the phragmoplastin protein
  • IPTG lsopropyl-B-D-thiogalactopyranoside
  • the construct was then transformed into E. coli Lys-s cells
  • the cells were grown m uria-Bertani (LB) medium to identify transformants . Expression of the phragmoplastin protein was induced by adding 0 5 mM IPTG to the medium of the transformed cells
  • Phragmoplastin protein was purified from a solubilized cell pellet by chromatography on a Nickel (Ni) chelate column obtained from Invitrogen. The Ni- affinity chromatography was conducted under non- denaturmg conditions according to the manufacturer's instructions The protein eluted from the Ni column was further purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) The gel-purified phragmoplastin protein was cut out from the polyacrylamide gel and ground m liquid nitrogen to provide the gel- pu ⁇ fied phragmoplastin. A single band of protein with an apparent molecular weight of
  • the molecular weight of the expressed protein results from fusion of a 35 ammo acid polyhistidme metal binding domain and a four ammo acid linker sequence to the N-termmus of the expressed phragmoplastin protein molecules.
  • the expressed protein also has the ability to hydrolyze GTP without the requirement for any other protein factors
  • phragmoplastin has intrinsic GTPase activity
  • the gel-purified phragmoplastin was directly injected into rabbits using conventional techniques
  • the animals were bled and the serum subjected to protein A gel chromatography as described m Harlow and Lan, "Antibodies," A Laboratory Manual. Cold Spring Harbor Laboratory 1988 to provide anti-phragmoplastin antibodies
  • Assaying for Phragmoplastin at Different Stages of the Cell Cycle The anti- soybean antiphragmoplastm antibodies prepared as described above were used to assay for phragmoplastin tobacco BY- 2 cells synchronized at S phase, metaphase, and cytokinesis by the methods of Asada and Shibaoka as described m J.
  • polyclonal and monoclonal anti-phragmoplastin antibodies enable one to monitor the distribution of phragmoplastin m dividing plant cells and to examine cell plate development.
  • the antibodies are used m an immunolabelmg method
  • the antibodies are used to immunolabel dividing cells, such as for examples cells that have been obtained from root tips or plant cell cultures. During the immunolabelmg procedure, the cells are fixed, permeablized and incubated with the anti-phragmoplastm antibody.
  • the cell walls are removed prior to incubation with the anti-phragmoplastm antibody to improve labeling
  • the cells are incubated with a second antibody which binds to the anti- phragmoplastm antibody and which carries a fluorescent or chemilummescent tag.
  • the cells are then visualized under a microscope and the distribution of the immunolabeled phragmoplastin tne cell determined.
  • Example 1 Immunolabelmg Cell Plates m Dividing Cells.
  • the plant tissues were then digested m a solution containing 2% cellulase from Calbiochem, 1% macerozyme R-10 from Ykult, Honsha, Japan, 0.5% pectolyase Y23 from Karlan m washing buffer for 15 minutes. After rmsmg m PHEM buffer, the plant tissues were squashed between coverslips to release the individual cells.
  • the cells were air dried for 10 minutes and then permeablized with 0 5% Triton X-100 m PHEM for 5 minutes
  • the phragmoplastin m the cells was then immunolabeled using affm ty-purifled anti- phragmoplastin IgG as the first antibody and fluorescem isothiocyanate (FITC) -conjugated anti-rabbit goat IgG as a second antibody.
  • FITC fluorescem isothiocyanate
  • Pre- immune serum was used as the first antibody
  • control group DNA was stained w th 4 ' 6 ' -d ⁇ am ⁇ dmo-2-phenylmdole (DAPI) at a concentration of 10 ⁇ g/ml for 5 minutes at room temperature.
  • DAPI fluorescem isothiocyanate
  • phase contrast microscopy did not enable visualization of the cell plate anaphase When the cells reached telophase the cell plate could De distinguished m the phase contrast image.
  • Intact cells from soybean root tips were treated as described above m example 1 except that the permeabilized cells were also incubated with mouse anti-tubulm IgG, obtained from Amersham and with tetramethylrhodamme isothiocyantate (TRITC) -conjugated goat anti-mouse IgG from Sigma to stain the microtubules.
  • the double- labeled cells were visualized m a confocal microscope equipped with dual excitation/emission filter sets for both FITC and TRITC. Photographs of the cells were taken m phase contrast, and using white light and blue light and appropriate filters for FITC, TRITC and DAPI fitted to a Zeiss microscope.
  • Three-day-old tobacco BY-2 cells were fixed m 4% paraformaldehyde m 60 mM Pipes, 25 mM Hepes, 10 mM EGTA, and 2 mM MgC12 , pH 7 0 (PHEM) for 1 hours at room temperature and then washed on one layer of Miracloth with buffer containing 10 mM MES , pH 5.7, 30 mM CaCl 2 , 0.1% BSA, 5 mM ⁇ -mercaptoethanol and 0.4 M mannitol Cells were then digested for 15 minutes a solution containing 1% cellulase from Calbiochem, San Diego, CA and 0.1 % pectolyase Y23 m washing buffer.
  • Transgenic cells which express a chimeric phragmoplastin protein that can be visualized n living cells by microscopy are prepared by transforming plant cells with a vector, such as for example the plant vector Agrrobactenurn which contains a DNA molecule that encodes phragmoplastin linked at its N-termmus or C-termmus to a luminescent polypeptide
  • a vector such as for example the plant vector Agrrobactenurn which contains a DNA molecule that encodes phragmoplastin linked at its N-termmus or C-termmus to a luminescent polypeptide
  • cultured plant cells with a vector containing a DNA molecule having a coding sequence for phragmoplastin protein linked to a coding sequence for the green fluorescence protein (GFP) as described below to provide transgenic cell lines that express the chimeric phragmoplastin protein, GFP-phragmoplastin
  • GFP green fluorescence protein
  • the plasmid pBI-35S-mGFP which contains a nucleotide sequence encoding the GFP was obtained from J Haesoloff, MRC Laboratory,
  • a plasmid containing the GFP encoding sequence can also be obtained from Clonetech Labs, Palo Alto, CA
  • the plasmid was amplified using standard techniques and then the GFP coding region was cloned into plasmid pUC-GFP44 obtained from Dr Z Yang, Ohio State University, Columbus, OH
  • the GFP coding region was then excised and ligated into the Xbal-Sacl sites of PBI221-JR, also obtained from Dr Z Yang, to provide plasmid pE-GFP44, which contained a Cauliflower mosaic virus 35S promoter, a GFP coding sequence with a Bglll site at the C-termmal end, and a nopalme synthase terminator
  • the Bglll-Sacl fragment of phragmoplastin coding sequence as shown SEQ ID NO 5 was removed from plasmid pEPDL12, prepared as described above, and cloned into the Bglll-
  • Plasmid pBI121-EGP contained a 35S promoter, a coding sequence for a GFP-phragmoplastm, and a nopalme synthase terminator Plasmid pBI121-EGP was transformed into Agrooacte ⁇ um tumefaciens LBA4404 using a treeze-thaw method
  • BY-2 cells were transformed using the procedure of An et al , "Binary Vectors", In Plant Molecular Biology Manual, pp A3/1, 1992
  • a five ml liquid culture of 3-day-old BY-2 cells was mixed with 100 ⁇ L of Agrobacterium tumefaciens LBA4404 harboring the plasmid pBI121-EGP
  • the cells were allowed to grow m Pet ⁇ plates at 25 °C without shaking for 2 days Cells were then filtered on one layer of Miracloth ana washed to remove most of the bacteria
  • the washed cells were then plated on solid Murashige and Skoog medium containing 300 ⁇ g kanamycm and 500 ⁇ g of carbenicillm Transgenic call usually emerged after 2-3 weeks
  • the calli were then transferred to new antibiotic-containing plates.
  • Transgenic cells (20 mg fresh weight) from different calli were digested for 20 minutes at room temperature m 200 ⁇ L of enzyme solution of 1% cellulase m 10 mM Mes, pH 5 7 and 0.38 M sorbitol . Cells were then cent ⁇ fuged for 2 minutes in a microcent ⁇ fuge at room temperature. The enzyme solution was removed and the cells were washed with 200 ⁇ L of washing solution containing 1 % Triton X-100 m PHEM. After pelleting, the cells were directly solubilized SDS sample buffer and processed for SDS-PAGE and protein gel blotting.
  • GFP-phragmoplastm which has a molecular weight of approximately 90 kDa as determined by SDS-gel electrophoresis .
  • About 50% of the kanamycm-resistant cell lines tested were found to express GFP-phragmoplastm at a level 2 to 5 fold higher than native phragmoplastin protein and three lines overexpressed the GFP-phragmoplastm at levels that were 5 to 10 fold higher than the levels of native phragmoplastin in untransformed BY-2 cells.
  • GFP-phragmoplastm is stable in the transgenic BY-2 cells. Even though a degradation product of the native phragmoplastin was found m most of the preparations, no degradation products of GFP-phragmoplastm were detected by protein gel blots analysis The degradation products of the native phragmoplastin were not detected if cell extract was made m the presence of protemase inhibitors .
  • the GFP-phragmoplastm was predominantly associated with the microsomal membrane fraction Fluorescence microscopy showed that the transgenic cells overexpressmg GFP-phragmoplastm at 2 to 10 fold higher than native phragmoplastin exhibited bright green fluorescence on the cell plate during cytokinesis, whereas no green fluorescence was observed m untransformed BY-2 cells Control BY-2 cells, which were transformed with GFP alone exhibited a green fluorescence throughout the cytoplasm but did not exhibit a defined fluorescent line in the cell plate region Thus, the targeting of the GFP-phragmoplastm to the cell plate is the function of the phragmoplastin part of the chimeric protein.
  • the development of the cell plate was examined m transgenic cells which were prepared as described above and which overexpress GFP-phragmoplastm to levels 5 to 10 X greater than the levels of native phragmoplastin m untransfor ed BY-2 cells. All transgenic cells were grown on solid medium or m liquid medium. Then 100 ⁇ L of liquid culture cells were mixed with 100 ⁇ L of medium m a culture well on a slide. The development of the cell plate the cells was examined over time n an individual cell by visualizing the GFP- phragmoplastm fluorescence under an inverted epifluoresence microscope equipped with an FITC filter set and taking photographs of the cell at different stages in the cell cycle. To reduce photobleachmg, an excitation light was turned on only when photographs were taken.
  • the GFP-phragmoplastm appeared as a dense short fluorescent line m the center of the dividing cells
  • the GFP-phragmoplastm fluorescence appeared soon after the onset of anaphase and gradually increased m intensity forming a short line within 30 minutes.
  • the fluorescence m the center of the cell plate decreased while the intensity of the fluorescence at the margin of the cell plate increased.
  • a ring-like, annular fluorescence was observed m the cell.
  • the cell plate enlarged, there was a gradual decrease of the overall intensity of the GFP-phragmoplastm fluorescent ring. After the cell plate reached the parental cell wall, the green fluorescence gradually disappeared.
  • the fluorescence of GFP-phragmoplastm on the cell plate remained stable up to 2 hours.
  • the chimeric protein GFP-phragmoplastm follows the same pattern of distribution during cytokinesis as native phragmoplastin .
  • the GFP- phragmoplastm fluorescence was confined to the center of the phragmoplast proper During the later stage, when the cell plate extended beyond the phragmoplast proper, the GFP fluoroescence redistributed to the growing edge of the cell plate.
  • GFP- phragmoplastm like native phragmoplastin s associated with the cell plate.
  • the time for the cell plate to fuse with the prenatal cell wall was greater than m non-transformed cells and often resulted the formation of an oblique cell plate.
  • transgenic cells which overexpress a phragmoplastin protein fused to a luminescent marker permit observation of cell plate development m a living cell continuously and throughout the entire process of cytokinesis. Accordingly, such transgenic cell lines provide a research tool which avoids many of the problems that are encountered when conventional research tools are used to examine cell plate formation. For example, use of the transgenic cell lines eliminates the need to fix the cells, which can occasionally distort subcellular structures. Use of the transgenic cell line also avoids the lengthy staining steps which are often required to examine cell plate development.
  • transgenic cell line also permits examination of cell plate development a single cell, and as such, avoids any problems that may result from observing a dynamic process m a multitude of cells which appear to be m different stages of the cell cycle. Thus, it is less likely that short-lived steps m cell plate development will be overlooked when such transgenic cell lines are used to examine cell plate development.
  • Examining the Effect of Herbicides on Cell Plate Development In one embodiment, the effect of herbicides whether known herbicides or herbicides on cell plate development is examined using anti-phragmoplastm antibodies.
  • the antibodies are used an immunolabelmg method which commences with application of the herbicide to dividing plant cells
  • various concentrations of the herbicide are applied to determine the optimum concentration for inhibiting cell plate development.
  • the herbicide may be applied to a whole plant or plant segment that comprises dividing cells, it s preferred that the herbicidal compound be added directly to the medium of plant cell cultures
  • the use of plant cell cultures eliminates the step of dissociating the cells of the growing plant
  • the use of plant cell cultures allows synchronization of the dividing cells to the same stages of mitosis When applied tc synchronized cells, it is preferred that the herbicidal compound be applied before anaphase. Thereafter, the cells typically are fixed, permeablized and incubated with the anti-phragmoplastm antibody
  • the cell walls are removed prior to incubation with the anti-phragmoplastm antibody to enhance labeling with the antibody
  • the cells are then incubated with a second antibody which binds to the anti-phragmoplastm antibody and which carries a fluorescent or chemilummescent tag.
  • the cells are then visualized under a microscope and the distribution of the immunolabeled phragmoplastin the cell is determined to identify herbicides which inhibit cell plate formation and development and to characterize the stages cell plate development where such herbicides exert their effect.
  • the herbicide is applied to transgenic plant cells that express a chimeric phragmoplastin protein that comprises phragmoplastin fused to a luminescent peptide or protein tag. Thereafter, the distribution of the chimeric protein withm the living cell is continuously monitored to determine the effect of the herbicide on cell plate development To permit easy visualization of the chimeric protein, it is preferred that the transgenic cells overexpress the chimeric protein to levels that are 5 to 10 fold higher than the level of native phragmoplastin.
  • the transgenic cells are synchronized prior to treatment with the herbicide.
  • the herbicide is applied to the cells at various stages m the cell cycle, including metaphase, anaphase, and telophase.
  • the distribution of the chimeric protein is monitored over time m living cells that have not been fixed or permeabilized
  • the distribution of the chimeric protein is monitored using microscopy, such as for example, epifluorescence microscopy as described m examples 3 and 4 or wide-field microscopy followed by image deconvolution as described by Gens J.S.
  • taxol The effect of taxol on cell plate development was determined as described m example 4 for caffeine, except that 10 ⁇ L of taxol rather than caffeine was added to the medium to a final concentration 50 mM

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Abstract

La présente invention concerne de nouveaux moyens et méthodes permettant d'identifier des herbicides et des herbicides potentiels nuisant à la formation et au développement de plaques cellulaires. On a découvert une nouvelle protéine, la phragmoplastine, qui est associée à des vésicules de membrane de plaque cellulaire durant la cytocinèse. En visualisant la phragmoplastine, on peut examiner le développement de la plaque cellulaire, en particulier en réponse à l'action d'herbicides ou d'herbicides potentiels. Une méthode de visualisation de phragmoplastine consiste à faire appel à des techniques immunocytochimiques avec des anticorps anti-phragmoplastine. Une autre méthode de visualisation de phragmoplastine consiste à faire appel à des cellules transformées avec une molécule d'ADN codant une protéine de phragmoplastine chimérique composée de phragmoplastine et d'une protéine ou d'un marqueur luminescent, enchaîné à la protéine de phragmoplastine. On visualise la phragmoplastine dans de telles cellules en examinant les cellules dans les conditions dans lesquelles le marqueur devient visible comme par microscopie par fluorescence. La présente invention concerne également des molécules d'ADN isolées codant une phragmoplastine, et des cellules transformées avec un ADN codant une phragmoplastine, en particulier un ADN codant des protéines de phragmoplastine chimériques.
PCT/US1998/002858 1997-02-13 1998-02-12 Phragmoplastine et methodes d'examen du developpement de plaques cellulaires Ceased WO1998035687A1 (fr)

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WO2002024914A1 (fr) * 2000-09-25 2002-03-28 Oji Paper Co.,Ltd. Modification du composant de parois cellulaires vegetales et methode permettant de reguler la differentiation du developpement

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EP1107005A1 (fr) * 1999-12-07 2001-06-13 Boehringer Ingelheim International GmbH Méthode pour l'identification d'inhibiteurs de cytokinèse
WO2001042794A3 (fr) * 1999-12-07 2001-11-15 Boehringer Ingelheim Int Procede d'identification des inhibiteurs de la cytocinese
WO2002024914A1 (fr) * 2000-09-25 2002-03-28 Oji Paper Co.,Ltd. Modification du composant de parois cellulaires vegetales et methode permettant de reguler la differentiation du developpement

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